ABSTRACT
<p><b>OBJECTIVE</b>To understand the molecular basis for a potential reaction mechanism and develop novel antibiotics with homology modeling for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS).</p><p><b>METHODS</b>The genetic engineering technology and the composer module of SYBYL7.0 program were used, while the HMGS three-dimensional structure was analyzed by homology modeling.</p><p><b>RESULTS</b>The mvaS gene was cloned from Streptococcus pneumoniae and overexpressed in Escherichia coli from a pET28 vector. The expressed enzyme (about 46 kDa) was purified by affinity chromatography with a specific activity of 3.24 micromol/min/mg. Optimal conditions were pH 9.75 and 10 mmol/L MgCl2 at 37 degrees C. The V(max) and K(m) were 4.69 micromol/min/mg and 213 micromol/L respectively. The 3D model of S. pneumoniae HMGS was established based on structure template of HMGS of Enterococcus faecalis.</p><p><b>CONCLUSION</b>The structure of HMGS will facilitate the structure-based design of alternative drugs to cholesterol-lowering therapies or to novel antibiotics to the Gram-positive cocci, whereas the recombinant HMGS will prove useful for drug development against a different enzyme in the mevalonate pathway.</p>
Subject(s)
Amino Acid Sequence , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Bacterial , Physiology , Hydroxymethylglutaryl-CoA Synthase , Chemistry , Genetics , Metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Streptococcus pneumoniae , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the efficacy of gene therapy with human vascular endothelial growth factor-c (VEGF-C) on obstructive lymphedema.</p><p><b>METHODS</b>Two animal models of lymphedema were created: one in the right hind limb of adult New Zealand white rabbits and the other in SD mouse tail. Each model was randomly divided into two groups to receive intradermal injection of either VEGF-C gene (experimental group), or saline(control group). In rabbit model, the volume change of affected limb was measured. In mouse model, biopsy was performed after 3 weeks treatment to detect the expression of VEGF-C mRNA and proteins. The lymphagenesis was evaluated by immunohistochemical examination with lymphatic endothelium hyaluronan receptor antibody.</p><p><b>RESULTS</b>The volume of the affect rabbit limb decreased by (24.40 +/- 1.08) ml in experimental group, compared with (5.80 +/- 1.92) ml in control group (P = 0.0001). The expression of VEGF-C mRNA and protein increased markedly in experiment group, but not in controls. More lymphatic vessels with large caliber were seen in experiment group (P = 0.0004).</p><p><b>CONCLUSIONS</b>VEGF-C gene therapy may alleviate or treat lymphedema by inducing lyphmangiogenesis.</p>
Subject(s)
Animals , Humans , Rabbits , Rats , Disease Models, Animal , Gene Transfer Techniques , Genetic Therapy , Lymphedema , Therapeutics , RNA, Messenger , Genetics , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor C , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the effect of adeno-BMP7 transfection on the biology of bone marrow stromal cells (BMSCs).</p><p><b>METHODS</b>Bone marrow was obtained from the goat. The BMSCs were isolated and cultured at the second passage. Once the cells attached and formed a monolayer with 70%-80% confluency, adeno-BMP7 (M.O.I. = 100) was added to the cells. After three days, calcium node was examined with staining; cell-coral compound was replanted subcutaneously.</p><p><b>RESULTS</b>With adeno-BMP7 transfection, BMP7 expression was detected with Western-blot; big calcium nodes were observed with staining. New bone formation was enhanced, which was evaluated by X-ray and histological examinations.</p><p><b>CONCLUSIONS</b>BMSCs transfected with adeno-BMP7 show much stronger osteogenic ability.</p>